The orderly segregation of chromosomes at mitosis requires precise control over the activity of kinetochores, the structures that anchor and move chromosomes along the microtubules of the mitotic spindle. Kinetochores are comprised of centromeric DNA and set of associated proteins, whose identities and functions are largely unknown. The goal of this work is to identify the proteins in the Saccharomyces cerevisiae kinetochore that mediate binding to and translocation along microtubules and to determine the functions and modes of action of these proteins. This will be accomplished by reassembling yeast kinetochores in vitro, first with crude extracts and then with progressively more purified components. The biochemical properties and composition of reassembled kinetochores will be determined and, using genetics, these properties will be related to the functions of kinetochores in vivo. The assembly of S. cerevisiae kinetochores in vitro is made possible by techniques recently developed by Drs. Sorger and Hyman. To determine the contribution of a recently identified complex of kinetochore proteins (CBF3) and of other as-yet unidentified proteins to the microtubule binding activity of kinetochores, the following specific experiments will be performed: The cell cycle-dependent regulation of the binding of kinetochores to microtubules will be investigated and the contribution of CBF3 and microtubule binding factors to this regulation will be determined. Wild-type and mutated CBF3 proteins will be expressed and the CBF3 domains involved in binding to DNA and in assembling microtubule-binding kinetochore complexes will be determined. The proteins that interact with CBF3 and bind kinetochores to microtubules will be characterized and isolated by biochemical fractionation of yeast extracts. Elucidation of the mechanism of kinetochore action will be essential to an understanding of why chromosomes are transmitted faithfully in normal cells but are frequently lost during the division of tumor cells. S. cerevisiae kinetochores have been chosen for study because their relative simplicity makes them particularly well suited to biochemical analysis. However, recent work on the cell cycle suggests that the mechanisms of mitosis are conserved among eucaryotes and thus, that genes and methods derived from the study of yeast will be useful in future studies of chromosome segregation and genome instability in human cells.